Dynamic incubator system and method

ABSTRACT

A dynamic incubator system and method for mammalian cells. The dynamic incubator system includes an incubator and a programming device communicatively coupled to the incubator. The programming device includes a memory, one or more processors, a display, and an input mechanism. The programming device is adapted to enable at least one preset temperature and gas concentration sequence to be one or more of designed for the interior of the incubator, saved to the memory of the programming device and/or selected for implementation within the interior of the incubator. The at least one temperature and gas concentration sequence includes programmed changes to the temperature and a CO2 gas concentration. A purging mechanism is coupled to the incubator and releases a portion of a gas concentration to enable rapid changes in the temperature and gas concentration in the incubator.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/816,624 filed Mar. 11, 2019. The entire contents of this applicationare incorporated herein by reference in their entirety.

TECHNICAL FIELD

This disclosure relates to cell incubator systems and methods, and inparticular, to a system and method for dynamically incubating cells,such as eukaryotic cells or mammalian cells.

BACKGROUND

Mammalian cells are typically grown under carefully controlledconditions. These conditions vary by cell type, but generally consist ofa suitable flask or dish, with a substrate or medium that suppliesessential nutrients (amino acids, carbohydrates, vitamins, minerals),growth factors, hormones, and regulates pH, osmotic pressure, andtemperature. Cells are grown and maintained at an appropriatetemperature and gas mixture (typically, 37° C., 5% CO₂ for mammaliancells) in a cell incubator. Commercially available cell incubators aredesigned to keep cells at a constant temperature and gas mixture.

It is generally known that when culturing mesenchymal stem cells, andother mammalian cells, some cellular processes are altered by changes intemperature, and changes in both O₂ and/or CO₂ concentration. Indeed, ithas been reported that increased temperature can alter the secretome ofmesenchymal stem cells (https://www.ncbi.nlm.nih.gov/pubmed/30389270)and that hypoxia improves mesenchymal stem cell osteopotency(https://www.ncbi.nlm.nih.gov/pubmed/30537732).

It is also generally known that cells within the human body do notremain at a constant temperature, experience variable concentrations ofO₂ and CO₂, and are subjected to physiological stresses. Not only dothese stresses alter signaling mechanisms in cells, but populations ofcells take advantage of these stressors to improve the overall healthand viability of the cell population. That is, after exposing cells tochanges in temperature and/or gas concentrations, the resultingpopulation of cells are healthier and more viable. However, standardincubators enable different settings in temperature or gasconcentrations, but they do not enable programmable rapid changes inthese conditions. Standard incubators have a significant deficiency inthat they do not enable the design, control, and implementation ofdynamic temperature and gas concentrations (and consequently pHvariations) over time, and thus maintain mammalian cells in conditionsdistinct from in vivo physiological conditions, which are notsteady-state. Indeed, constant temperature and gas concentrations instandard mammalian cell incubation disregards the possibility thatnatural mammalian cellular function requires dynamic changes intemperature and gas concentrations, and that cellular health andphysiologically germane cellular dynamics rely upon intermittent changesin temperature and gas concentrations.

SUMMARY OF THE DISCLOSURE

In accordance with one exemplary aspect of the present disclosure, adynamic incubator system for cells, such as mammalian cells, comprisesan incubator having a housing with an interior adapted to containmammalian cells and a display for providing one or more of an actualtemperature and a gas concentration of the interior of the incubator. Aprogramming device is communicatively coupled to the incubator via acommunication network. The programming device includes a memory, one ormore processors, a display, and an input mechanism. The programmingdevice is also adapted to enable at least one preset temperature and gasconcentration sequence to be one or more of designed for the interior ofthe incubator, saved to the memory of the programming device and/orselected for implementation within the interior of the incubator. The atleast one temperature and gas concentration sequence includes programmedchanges to the temperature and one or more of a CO₂ gas concentration oran O₂ gas concentration in the interior of the incubator. In addition, apurging mechanism is coupled to the incubator and the programmingdevice. The purging mechanism is adapted to release one or more of: (1)a portion of a gas concentration disposed within the interior of theincubator; or (2) an amount of water disposed in an internal tank of theincubator or a water jacket disposed on the incubator to enable rapidchanges in the temperature and one or more of gas concentration in theinterior of the incubator or the amount of water in the internal tank orthe water jacket.

In accordance with another exemplary aspect of the present disclosure, amethod of dynamically incubating cells, such as mammalian cells,comprises one of designing or selecting at least one temperature and gasconcentration sequence for an interior of an incubator via a programmingdevice communicatively coupled to the incubator. The method furthercomprises executing the at least one temperature and gas concentrationsequence within the interior of the incubator. The at least onetemperature and gas concentration includes programmed changes to thetemperature and one or more of an CO₂ gas concentration or an O₂ gasconcentration in the interior of the incubator after designated periodsof time. Lastly, the method further includes enabling rapid changes intemperature and gas concentrations within the interior of the incubatorvia a purging mechanism coupled to the incubator and the programmingdevice.

Additional optional aspects and features are disclosed, which may bearranged in any functionally appropriate manner, either alone or in anyfunctionally viable combination, consistent with the teachings of thedisclosure. Other aspects and advantages will become apparent uponconsideration of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

It is believed that the disclosure will be more fully understood fromthe following description taken in conjunction with the accompanyingdrawings. Some of the drawings may have been simplified by the omissionof selected elements for the purpose of more clearly showing otherelements. Such omissions of elements in some drawings are notnecessarily indicative of the presence or absence of particular elementsin any of the example embodiments, except as may be explicitlydelineated in the corresponding written description. Also, none of thedrawings are necessarily to scale.

FIG. 1A is front perspective view of a dynamic incubator systemaccording to one aspect of the present disclosure;

FIG. 1B is a block diagram of a portion of the dynamic incubator systemof FIG. 1A;

FIG. 2A is another front perspective view of the dynamic incubatorsystem of FIG. 1A, with a purging mechanism according to one aspect ofthe present disclosure;

FIG. 2B is a front perspective view of the dynamic incubator system ofFIG. 2A including another purging mechanism according to another aspectof the present disclosure;

FIG. 2C is a front perspective view of the dynamic incubator system ofFIG. 2B according to another aspect of the present disclosure;

FIG. 3 is a rear perspective view of the dynamic incubator system ofFIG. 1A;

FIG. 4 is a close-up view of a portion of the dynamic incubatory systemof FIG. 1A;

FIG. 5 is an exemplary flow chart depicting a method of one aspect ofthe present disclosure;

FIG. 6 is a graph depicting the results of a cell proliferationexperiment using the dynamic incubator system of the present disclosure;

FIG. 7 is a series of photographs depicting the results of a cellmorphology experiment involving the dynamic incubator system and methodsof the present disclosure;

FIG. 8 is another graph depicting the results of another cellproliferation experiment using the dynamic incubator system and methodsof the present disclosure;

FIG. 9 is a graph depicting the results of a cell senescence experimentusing the dynamic incubator system and methods of the presentdisclosure;

FIG. 10 is another graph depicting the results of an interleukin-6 cellproduction experiment using the dynamic incubator system and methods ofthe present disclosure; and

FIG. 11 is another graph illustrating the results of another cellproliferation experiment using the dynamic incubator system and methodsof the present disclosure.

DETAILED DESCRIPTION

Generally, a system and method of dynamically incubating cells, such asmammalian cells, is disclosed. Specifically, in vitro cell expansion canbe greatly improved by adding dynamic changes in temperature, gasconcentrations, and/or mechanical stress to the incubation system. Adynamic incubation system includes an incubator and a programming devicecommunicatively coupled to the incubator. The programming device enableschanges in temperature and gas concentrations (e.g., CO₂ and O₂) to bepreprogrammed, such as having at least one preset temperature and gasconcentration sequence designed and/or saved via the programming device,allowing cells to be grown in dynamic conditions. In addition, a purgingmechanism, such as an air pump with an air lock, is also communicativelycoupled to both the incubator and the programming device. The purgingmechanism releases a portion of the gas concentration within theincubator to enable rapid changes in the temperature and gasconcentrations within the incubator, as explained more below. Soconfigured, growing mammalian cells in such dynamic temperature and gasconcentrations according to specific sequences results in reduced celldoubling time, reduced cellular senescence, and reduced inflammatorycytokine production when compared to cells grown at constant temperatureand gas concentrations.

Referring now to FIGS. 1A and 1B, a dynamic incubator system 10according to the present disclosure is depicted. The dynamic incubatorsystem 10 includes an incubator 12 having a housing 14 with an interior16 adapted to contain stem cells. A display 18 is disposed on an outsideportion of the housing 14 of the incubator 12, such as a central, frontoutside portion of the housing 14, as depicted in FIG. 1. Alternatively,the display 18 may be disposed on any other outside portion of thehousing 14 of the incubator 12 and still fall within the scope of thepresent disclosure. The display 18 provides one or more of an actualtemperature of the interior 16 of the incubator 12 or an actual gasconcentration, such as one or more of a CO₂ gas concentration and an O₂gas concentration, of the interior 16 of the incubator 12.

A programming device 20 is communicatively coupled to the incubator 12via communication network 21. As will be appreciated, the communicationnetwork 21 may be one of a wired or a wireless connection and fallwithin the scope of the present disclosure. As depicted in FIG. 1B, theprogramming device 20 includes a memory 22, one or more processors 24, adisplay 26, an input mechanism 28, at least one transmitter 30, and atleast one receiver 32. The programming device 20 is adapted to enable atleast one temperature and gas concentration sequence to be designed andpreprogrammed for the interior 16 of the incubator 12 via the inputmechanism 28, for example. Alternatively and/or additionally, theprogramming device 20 may be incorporated into the incubator housing 14itself using the incubator display mechanism 18 and input mechanism.Alternatively and/or additionally, the programming device 20 may alsoenable at least one preset temperature and gas concentration sequence tobe saved to the memory 22 of the programming device 20. In this manner,the at least one preset temperature and gas concentration sequence maybe displayed on the display 26, for example, and selected forimplementation within the interior 16 of the incubator 12 via one ormore of the display 26 or the input mechanism 28, as explained morebelow.

In addition, the at least one temperature and gas concentration sequenceincludes programmed changes to the temperature and a CO₂ gasconcentration in the interior 16 of the incubator 12. In anotherexample, the at least one temperature and gas concentration sequence mayinclude programmed changes to the temperature, a CO₂ gas concentration,and an O₂ gas concentration in the interior 16 of the incubator 12, asalso explained more below.

As further depicted in FIG. 1B, the dynamic incubator system 10 mayfurther include a user control device 40. The user control device 40 maybe communicatively coupled to at least one of the programming device 20and the communication network 21 and used to activate the programmingdevice 20, for example. In addition, the user control device 40 includesa memory 42 and one or more processors 44. Further, the user controldevice 40 may also include a display 40, an input 42, at least onetransmitter 50, and at least one receiver 52. So configured, a user mayremotely activate the programming device 20 via the user control device40 to control the temperature and gas concentration conditions withinthe interior 16 of the incubator 12.

Moreover, the programming device 20 may include a personal computer,such as a laptop computer, as depicted in FIG. 1A. However, as will beappreciated, the programming device 20 may take the form of variousother electronic devices capable of the implementing the same functionsas the personal computer and still fall within the scope of the presentdisclosure. For example, the programming device 20 may alternativelyand/or additionally include any one or more of a smart phone, a tablet,an e-reader or any other similar electronic device, or the programmingdevice 20 may be incorporated into the incubator 12 itself.

Referring now to FIG. 2A, the dynamic incubator system 10 furtherincludes a purging mechanism 60. In some examples, the purging mechanismis a pump, such as an air pump. However, the purging mechanism 60 mayalternatively and/or additionally take the form of various other devicesand/or machines capable of implementing the functions of the purgingmechanism 60 described in more detail below and still fall within thescope of the present disclosure. In FIG. 2A, the purging mechanism 60includes an air pump having an air inlet tube 62 and an air outlet tube64. As further depicted, the purging mechanism 60 is alsocommunicatively coupled to the programming device 20 via a communicationnetwork, such as the communication network 21, which may be one or moreof a wired connection or a wireless connection. The purging mechanism 60and the coupled programming device 20 may be contained within thehousing 14 of the incubator 12.

Referring now to FIG. 2B, another purging mechanism 60 that mayalternatively or additionally be used with the dynamic incubator system10 is depicted. In this example, the purging mechanism 60 is a waterpump 60 a that cycles water from an internal water tank 65, disposedwithin the interior 16 of the incubator 12, to an external water tank 67and includes a water inlet tube 62 a and a water outlet tube 64 a.Specifically, the water inlet tube 62 a is coupled to the internal watertank 65, and the water outlet tube 64 a is coupled to the external watertank 67. The water pump 60 a may more efficiently change the temperatureof the interior 16 of the incubator 12. In one example, the externalwater tank 67 may heat or cool the water to better function as a heatsource or heat sink, respectively, when the water from the water pump 60a is introduced into the internal water tank 65 in the interior 16 ofthe incubator 12.

In another example, and instead of the internal water tank 65 disposedwithin the incubator 12, the water pump 60 a may exchange water from theexternal water tank 67 into a water jacket 69. The water jacket 69surrounds the interior 16 of the incubator 12 and is disposed on anoutside area of the housing 14, as depicted in FIG. 2C. In this example,the water jacket 69 extends around at least a part of the interior 16 ofthe incubator 12 and upwardly extends to an approximate mid-way portionand/or center area of the housing 14 of the incubator 12. However, itwill be appreciated that the water jacket 69 may extend up to a top areaof the housing 14 and/or extend to any other point or area along thelength of the housing 14 of the incubator 12 and still fall within thescope of the present disclosure.

Alternatively, both the water pump 60 a and the water jacket 69 may moregenerally be a fluid pump 60 a and a fluid jacket 69, each of which usesa heat transfer liquid other than water. For example, any other fluidaside from water, such as oil, synthetic hydrocarbon, silicon basedfluids, molten salts, molten metals, and various gases including watervapor, nitrogen, argon, helium and hydrogen, may alternatively be used(instead of water) and still fall within the scope of the presentdisclosure.

Referring now to FIG. 3, a rear view of the dynamic incubator system 10is depicted. A rear portion of the housing 14 is depicted and includes aport 63. The air inlet tube 62 and the air outlet tube 64 of the purgingmechanism 60 are both coupled to the port 63 on the housing 14 of theincubator 12. In addition, the outlet tube 64 is further connected to anair lock assembly 66. The air lock assembly 66 prevents loss of aportion or some of the gas concentration in the interior 16 of theincubator 12 when the purging mechanism 60 is not operating, forexample. In some embodiments, the air lock assembly 66, the air inlettube 62 and the air outlet tube 64 of the purging mechanism 60 may beincorporated in the housing 14 of the incubator 12.

The dynamic incubator system 10 may further include a module stored inthe memory 22 of the programming device 60. The module is executable bythe at least one processor 24 of the programming device 60 to set afirst temperature T1 of the interior 16 of the incubator 12, set a firstCO₂ gas concentration of the interior 16 of the incubator 12 to a valueG1, and then maintain the temperature T1 and the CO₂ gas concentrationat the value G1 for a time t1. The module may then further set a secondtemperature T2 of the interior 16 of the incubator 12 and set a CO₂ gasconcentration of the interior 16 of the incubator 12 to a value G2. Themodule may then operate the purging mechanism 60 for a time tp to purgea portion of the gas concentration of the interior 16 of the incubator12. Lastly, the module maintains the second temperature T2 and CO₂ gasconcentration at the value G2 for a time t3.

In one example temperature and gas concentration sequence, thetemperature T1 is 37 degrees C., the CO₂ gas concentration value G1 is5% CO₂, the time t1 is about 8 hours, the temperature T2 is 38 degreesC., the CO₂ gas concentration value G2 is 5.5% CO₂, the time tp is about1 minute, and the time t2 is about 30 minutes.

In another example temperature and gas concentration sequence, thetemperature T1is 38 degrees C., the CO₂ gas concentration value G1 is 6%CO₂, the time t1 is about 60 minutes, the temperature T2 is 37 degreesC., the CO₂ gas concentration value G2 is 5% CO₂ , the time tp is about1 minute, and the time t2 is about 8 hours.

In yet another example temperature and gas concentration sequence, thefirst temperature T1 is 36 degrees C., the first CO₂ gas concentrationvalue G1 is 5.5% CO₂, the time t1 is about 120 minutes, the secondtemperature T2 is 37 degrees C., the second CO₂ gas concentration valueG2 is 5% CO₂ , the time tp is about 1 minute, and the time t2 is about20 hours.

In this example, the module of the programming device 20 is furtherexecutable by the processor 24 to: set a O₂ gas concentration level to avalue G3 after the first temperature T1and the first CO₂ gasconcentration value G1 are set, and maintain each of the firsttemperature T1, the second CO₂ gas concentration value G1, and the O₂gas concentration value G3 for the time t1. In addition, the module thensets the CO₂ gas concentration value to a value G4 and the O₂ gasconcentration value to a value G5, and then maintains the CO₂ gasconcentration value to a value G4 and the O₂ gas concentration value toa value G5 for a period of time t1. Still further, the module of theprogramming device 20 is further executable by the processor 24 to setthe O₂ gas concentration value to a value zero before the purgingmechanism is operated for a time tp. In this example, the at least oneor more of the O₂ gas concentration value G3 is 18% O₂, the CO₂ gasconcentration value G4 is 6% CO₂, and the O₂ gas concentration valueG5is 15% O₂.

While several example temperature and gas concentration sequences areprovided above, it will be understood that various other temperature andgas concentration sequences may alternatively be designed and/orimplemented and still fall within the scope of the present disclosure.For example, one or more of the example temperatures T1, T2 and theexample gas concentrations G1, G2, G3, G4, and G5 may be many othervalues. In one example, the temperature values T1 and T2 may change,while the gas concentration values G1-G5 remain the same. In anotherexample, any one of the gas concentration values G1-G5 may change and bedifferent from one the examples provided above, while the temperaturevalues T1 and T2 remain the same. Further, both the temperature valuesT1 and T2 may change and any one of the gas concentration values G1-G5may likewise change and still affect dynamic incubation of the cells.Any of such variations still fall within the scope of the presentdisclosure.

Referring now to FIG. 4, a close-up view of the display 18 disposed onthe housing 14 of the incubator 12 is depicted. In this example, anactual gas concentration of a CO₂ gas concentration disposed within theinterior 16 of the incubator 12 over a period of time is displayed.Alternatively and/or additionally, the display 18 may provide an actualtemperature within the interior 16 of the incubator over a period oftime. Further, the display 18 may also list and/or display at least onelog of a previous actual temperature and a previous actual gasconcentration over a period of time. Said another way, listings ofprevious actual temperature and gas concentration ranges and/orsequences may be displayed on the display 18 of the dynamic incubator12.

Referring now to FIG. 5, a flowchart of an example method of dynamicallyincubating stem cells 100 is depicted. The method 100 may beimplemented, in whole or in part, on one or more devices or systems suchas those shown in the dynamic incubator system 10 of FIGS. 1-4. Themethod 100 may be saved as a set of instructions, routines, programs ormodules on a memory, such as the memory 22 of the programming device 20(FIG. 2), and may be executed by one or more processors, such as theprocessor 24 (FIG. 2) of the programming device 20.

The method 100 begins in block 102 when a user activates the programmingdevice 20, such as by directly interacting with the programming device20 or by remotely operating the programming device 20 via the usercontrol device 40 (FIG. 2). Next, in block 104, the user may design,such as program, at least one temperature and gas sequence for theinterior 16 of the incubator 12. Alternatively, in block 106, the usermay select at least one preprogrammed (e.g., preset) temperature and gasconcentration sequence for the interior 16 of the incubator 12. Morespecifically, a listing of several preprogrammed temperature and gasconcentration sequences, which have been saved to the programming device20, may be displayed on the display 26 of the programming device 20 uponactivation. After being displayed, the user may select one of thepreprogrammed temperature and gas concentration sequences for executionwithin the interior 16 of the incubator 12.

However, if the user designs the temperature and gas concentrationsequence (block 104), the user then selects a first temperature T1, afirst gas concentration level G1, and a first period of time t1 at whichthe temperature T1 and the gas concentration G1 will cycle in theinterior 16 of the incubator 12 in block 108. The first temperature T1,the first gas concentration level G1, and the first period of time t1may be selected via the input 28 (FIG. 2) of the programming device 20,for example. In block 110, the user then selects a second temperatureT2, a second gas concentration level G2, and a second period of time t2at which the second temperature T2 and the gas concentration level G2will cycle in the interior 16 of the incubator 12 after the first timet1 to help effect the dynamic incubation of the stem cells disposed inthe incubator 12. In this way, a preprogrammed temperature and gasconcentration sequence is designed. In block 112, the preprogrammedtemperature and gas concentration sequence is saved to the memory 22 ofthe programming device 20 and may be listed with other preprogrammedtemperature and gas concentration sequences displayed on the display 26of the programming device 20. Alternatively, the preprogrammedtemperature and gas concentration may be listed on the display 18 of theincubator 12 when the programming device 20 is incorporated into theincubator housing 14, as explained above.

In block 114, either the designed or the selected temperature and gasconcentration sequence is executed for implementation within theinterior 16 of the incubator 12. The temperature and gas concentrationsequence includes programmed changes to the temperature and one or moreof the CO₂ gas concentration or the O₂ gas concentration in the interior16 of the incubator 12 after designated periods of time, such as timest1 and t2.

In block 116, the purging mechanism 60, such as the air pump, purges(e.g., releases) an amount or a portion of gas concentration within theinterior 16 of the incubator 12 for period of time tp. In this way,rapid changes in the temperature and gas concentration within theinterior 16 of the incubator 12 are able to be effected, contributing tothe dynamic incubation of the stem cells. In another example, when thepurging mechanism 60 is the water pump 60 a, the water pump 60 a purges,e.g., cycles and/or releases, an amount of water in one of the internalwater pump 65 of the incubator 12 or the water jacket 69 surrounding atleast a portion of the interior 16 to the external water tank 67 for aperiod of time tp. In this way, rapid changes in the temperature withinthe interior 16 of the incubator 12 are again able to be effected, alsocontributing to the dynamic incubation of the stem cells.

In one example, executing the at least one temperature and gasconcentration sequence for the interior 16 of the incubator 12 of block114 comprises implementing the selected preprogrammed temperature andgas concentration sequence. This includes setting the first temperatureT1 and the first CO₂ gas concentration G1 and maintaining the firsttemperature T1 and the first CO₂ gas concentration G1 for a time t1. Theexecuting next includes setting the second temperature T2 and the secondCO₂ gas concentration G2 and purging a portion of the gas concentrationof the interior 16 of the incubator 12 via the purging mechanism 60 fora time tp. The executing step then also includes maintaining the secondtemperature T2 and the second CO₂ gas concentration G2 for a time t2.

In one example temperature and gas concentration sequence, setting thefirst temperature T1 and the first CO₂ gas concentration G1 of theinterior 16 of the incubator 12 is setting at the first temperature T1to 37 degrees C. and the first CO₂ gas concentration G1 to 5% CO₂ Inaddition, maintaining the first temperature T1 and the CO₂ gasconcentration G1 for a time t1 includes for the time t1 of about 8hours. Further, setting the second temperature T2and the second CO₂ gasconcentration to a value G2 includes setting the second temperature 38degrees C. and the second CO₂ gas concentration G2 to 5.5% CO₂. Stillfurther, purging a portion of the gas concentration of the interior 16of the incubator 12 via the purging mechanism 60 for a time tp, orpurging an amount of water in one of the internal water pump 65 of theincubator 12 or the water jacket 69 for a time tp, includes for a timeof about 1 minute. Lastly, maintaining the second temperature T2 and thesecond CO₂ gas concentration G2 for a time t2includes for a time t2 ofabout 30 minutes.

In another example temperature and gas concentration sequence, settingthe first temperature T1 and the first CO₂ gas concentration G1 includessetting the first temperature T1to 38 degrees C. and the first CO₂ gasconcentration G1 to 6% CO₂ In addition, maintaining the firsttemperature T1 and the CO₂ gas concentration G1 for a time t1 is for atime t1 of about 60 minutes. Further, setting the second temperature T2and the second CO₂ gas concentration to a value G2 includes setting asecond temperature to 37 degrees C. and the second CO₂ gas concentrationG2 to 5% CO₂. Still further, purging the portion of the gasconcentration of the interior 16 of the incubator 12 via the purgingmechanism 60 for a time tp, or purging an amount of water in one of theinternal water pump 65 of the incubator 12 or the water jacket 69 for atime tp, includes for a time of about 1 minute. Lastly, maintaining thesecond temperature T2and the second CO₂ gas concentration G2 for a timet2 includes for a time t2 of about 8 hours.

In still another example temperature and gas concentration sequence,setting the first temperature T1 and the first CO₂ gas concentration G1includes setting at the first temperature T1 to 36 degrees C. and thefirst CO₂ gas concentration G1 to 5.5% CO₂, In addition, maintaining thefirst temperature T1 and the CO₂ gas concentration G1 for a time t1 isfor a time t1 of about 120 minutes. Further, setting the secondtemperature T2 and the second CO₂ gas concentration to a value G2includes setting the second temperature to 37 degrees C. and the secondCO₂ gas concentration G2 to 5% CO₂. Still further, purging a portion ofthe gas concentration of the interior 16 of the incubator 12 via thepurging mechanism 60 for a time tp, or purging an amount of water in oneof the internal water pump 65 of the incubator 12 or the water jacket 69for a time tp, includes for a time of about 1 minute. Lastly,maintaining the second temperature T2 and the second CO₂ gasconcentration G2 for a time t2 includes for a time t2 of about 20 hours.

In this example, executing at least one temperature and gasconcentration sequence via the programming device 20 may furthercomprise setting an O₂ gas concentration level to a value G3 aftersetting the temperature T1 and the CO₂ gas concentration value G1 viathe programming device 20. In addition, executing may also includemaintaining each of the temperature T1, the CO₂ gas concentration valueG1, and the O₂ gas concentration value G3for the time t1 via theprogramming device 20 and setting the CO₂ gas concentration value to avalue G4 via the programming device 20. Further, executing may includesetting the O₂ gas concentration value to a value G5 via the programmingdevice 20, and maintaining the CO₂ gas concentration value to a value G4and the O₂ gas concentration value to a value G5 for a period of time t1via the programming device.

In this further example, executing may also include setting the O₂ gasconcentration value to zero before operating the purging mechanism 60for a time tp via the programming device 20. In addition, at least oneor more of the O₂ gas concentration value G3 is 18% O₂, the CO₂ gasconcentration value G4 is 6% CO₂, and the O₂ gas concentration value G5is 15% O₂.

While various temperature and gas sequences are described above, it willbe appreciated that several other temperature and gas sequences havingother selected first and second temperatures, first and second gasconcentrations, and cycling times may alternatively be used to affectthe dynamic incubation results and still fall within the scope of thepresent disclosure.

Referring now to FIG. 6, a graph illustrating the results of a cellproliferation experiment using the dynamic incubator system 10 of thepresent disclosure is depicted. In this experiment, cell counts ofmesenchymal stem cells after 144 hours of culture in controlledtemperature and gas concentration (constant 37° C. and 5% CO₂) arecompared with dynamic incubation using the dynamic incubator system 10(cycling 37° C. and 5% CO₂ for 8 hrs., then 38° C. and 5.5% CO₂ for 30min.). In the experiment, 35,000 human adipose MSCs were plated in a T75flask, containing 10 mL of α-MEM medium. The P-value is from t-testcompared to control.

Referring now to FIG. 7, a series of photographs illustrating theresults of a cell morphology experiment involving the dynamic incubatorsystem 10 and methods of the present disclosure is depicted. In thisexperiment, adipose mesenchymal stem cells after 168 hours of culture incontrolled, constant temperature and gas concentration (constant 37° C.and 5% CO₂), are compared to dynamic incubation using the dynamicincubator system 10 (cycling 37° C. and 5% CO₂ for 8 hrs., then 38° C.and 5.5% CO₂ for 30 min.). The experiment was conducted with thepresence of an investigational compound (PA20) that reduces MSCsenescence. In the experiment, 10,000 human adipose MSCs were plated ina T25 flask, containing 10 mL of α-MEM medium, with 5 uM PA20. Theresults show that MSCs in dynamic incubation have polar morphology,indicative of a non-differentiated/non-senescent state. The dynamicincubation appeared to have synergistic effect with PA20.

Referring to FIG. 8, another graph illustrating the results of anothercell proliferation experiment using the dynamic incubator system 20 andmethods of the present disclosure is depicted. In this experiment,adipose mesenchymal stem cells (A-MSCs) after 168 hours (P1) and anadditional 336 hours (P2) of culture in controlled temperature and gasconcentration (constant 37° C. and 5% CO₂), are compared to dynamicincubation using the dynamic incubator system 10 (cycling 37° C. and 5%CO₂ for 8 hrs., then 38° C. and 5.5% CO₂ for 30 min.). The experimentincluded an investigational compound (PA20) that reduces MSC senescence.For each passage (P1 and P2), 10,000 human adipose MSCs were plated in aT25 flask, containing 10 mL of a-MEM medium, with 5 uM PA20. Inaddition, cells from P1 were re-plated in P2, and P-values are fromt-test compared to respective P1 or P2 control. The results show dynamicincubation increases proliferation of A-MSCs, and further increasesproliferation in the presence of PA20.

Referring now to FIG. 9, a graph illustrating the results of a cellsenescence experiment using the dynamic incubator system 10 and methodsof the present disclosure is depicted. In this experiment, adiposemesenchymal stem cells after 168 hours of culture in controlledtemperature and gas concentration (constant 37° C. and 5% CO₂) arecompared with dynamic incubation using the dynamic incubator system 10(cycling 37° C. and 5% CO₂ for 8 hrs., then 38° C. and 5.5% CO₂ for 30min.). The experiment included an investigational compound (PA20) thatreduces MSC senescence. In addition, 10,000 human adipose MSCs wereplated in a T25 flask, containing 10 mL of α-MEM medium, with 5 uM PA20.β-galactosidase (a marker of cellular senescence) was measured by ELISAassay. Also, p-values are from t-test compared to respective control.The results show that β-galactosidase is reduced by the dynamicincubation.

Referring now to FIG. 10, another graph illustrating the results of aninterleukin-6 (IL-6) cell production experiment using the dynamicincubator system 10 and methods of the present disclosure is depicted.In this experiment, IL-6 is a pro-inflammatory cytokine, and adiposemesenchymal stem cells after 168 hours of culture in controlledtemperature and gas concentration (constant 37° C. and 5% CO₂) arecompared with dynamic incubation using the dynamic incubator system 10(cycling 37° C. and 5% CO₂ for 8 hrs., then 38° C. and 5.5% CO₂ for 30min.). The experiment included the presence of an investigationalcompound (PA20) that reduces MSC senescence. Further, 10,000 humanadipose MSCs were plated in a T25 flask, containing 10 mL of α-MEMmedium, with 5 uM PA20. The IL-6 was measured by ELISA assay. Theresults show that IL-6 is reduced by dynamic incubation. Notably,dynamic incubation reduced PA20-associated increase in IL-6. Thisindicates that dynamic incubation reversed the agonist effect of PA20 onMSC IL-6 production. In this experiment, p-values are from t-testcompared to respective P1 or P2 Control.

Referring now to FIG. 11, another graph illustrating the results ofanother cell proliferation experiment is depicted. In this experiment,rat 9L gliosarcoma cells after 72 hours of culture in controlledtemperature and gas concentration (constant 37° C. and 5% CO₂) arecompared with dynamic incubation using the dynamic incubator system 10(cycling 37° C. and 5% CO₂ for 8 hrs., then 38° C. and 5.5% CO₂ for 30min.). The experiment included 10 uM Fisetin. In addition, 100,000 9Lcells were plated in a T75 flask, containing 10 mL of a-MEM medium, with10 uM Fisetin. In addition, the medium was refreshed after 24 hourswithout Fisetin, and the cells were then cultured for a 48 additionalhours. The results show that the dynamic incubation increasesproliferation of 9L cells, and decreases cell numbers in the presence ofFisetin. P-values are from t-test compared to control.

In view of the foregoing, one of ordinary skill in the art willappreciate the following advantages of the system 10 and methods 100 ofthe present disclosure described above. For example, and as theforegoing experimental results show, growing cells, such as mammaliancells, in dynamic temperature and gas concentrations according tospecific sequences of temperature and gas concentrations (as describedabove) results in reduced cell doubling time, reduced cellularsenescence, and reduced inflammatory cytokine production when comparedto cells grown at constant temperature and gas concentrations. Moreover,when dynamic incubation is used in the presence of an investigatorycompound, the dynamic incubator system 10 synergistically increasedproliferation and reduced cell senescence, but counteracted thecompound's activation of IL-6 (an inflammatory cytokine), as explainedabove. Still further, the purging mechanism 60 allows for the quickpurging of the interior incubator atmosphere. This allows for quickchanges in the interior temperature and gas concentration to help effectthe dynamic incubation.

The following additional considerations apply to the foregoingdiscussion. Throughout this specification, plural instances mayimplement components, operations, or structures described as a singleinstance. Although individual operations of one or more methods areillustrated and described as separate operations, one or more of theindividual operations may be performed concurrently, and nothingrequires that the operations be performed in the order illustrated.Structures and functionality presented as separate components in exampleconfigurations may be implemented as a combined structure or component.Similarly, structures and functionality presented as a single componentmay be implemented as separate components. These and other variations,modifications, additions, and improvements fall within the scope of thesubject matter herein.

Some implementations may be described using the expression “coupled”along with its derivatives. For example, some implementations may bedescribed using the term “coupled” to indicate that two or more elementsare in direct physical or electrical contact. The term “coupled,”however, may also mean that two or more elements are not in directcontact with each other, but yet still co-operate or interact with eachother. The implementations are not limited in this context.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of elements is notnecessarily limited to only those elements but may include otherelements not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive or and not to an exclusive or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

In addition, use of the “a” or “an” are employed to describe elementsand components of the implementations herein. This is done merely forconvenience and to give a general sense of the invention. Thisdescription should be read to include one or at least one and thesingular also includes the plural unless it is obvious that it is meantotherwise.

Further, while particular implementations and applications have beenillustrated and described, it is to be understood that the disclosedimplementations are not limited to the precise construction andcomponents disclosed herein. Various modifications, changes andvariations, which will be apparent to those skilled in the art, may bemade in the arrangement, operation and details of the method and systemdisclosed herein without departing from the spirit and scope defined inthe appended claims.

1-15. (canceled)
 16. A method of dynamically incubating cells, themethod comprising: one of designing or selecting at least onetemperature and gas concentration sequence for an interior of anincubator via a programming device communicatively coupled to theincubator; executing the at least one temperature and gas concentrationsequence within the interior of the incubator, the at least onetemperature and gas concentration including programmed changes to thetemperature and a CO₂ gas concentration in the interior of the incubatorafter designated periods of time; and enabling rapid changes in one ormore of: (a) the at least one temperature and gas concentration withinthe interior of the incubator; or (2) a temperature within the interiorof the incubator via a purging mechanism coupled to the incubator andthe programming device.
 17. The method of claim 16, wherein designing atleast one temperature and gas concentration sequence for an interior ofan incubator comprises selecting a first temperature T1, a first CO₂ gasconcentration level G1, and a first period of time t1 at which thetemperature T1 and CO₂ gas concentration level G1 will cycle in theinterior of the incubator via the input of the programming device;selecting a second temperature T2, a second CO₂ gas concentration levelG2, and a second period of time t2 at which the temperature T2 and CO₂gas concentration level G3 will cycle in the interior of the incubatorto effect dynamic via the input of the programming device to form apreset temperature and gas concentration sequence; and saving the presettemperature and gas concentration sequence to the memory of theprogramming device.
 18. The method of claim 16, wherein selecting atleast one temperature and gas concentration sequence for an interior ofan incubator via a programming device comprises listing at least onetemperature and gas concentration sequence on the display of theprogramming device and selecting the at least one temperature and gasconcentration sequence for execution within the interior of theincubator via the input of the programming device.
 19. The method ofclaim 16, wherein executing at least one temperature and gasconcentration sequence for an interior of an incubator via a programmingdevice comprises: setting a first temperature T1 and a first CO₂ gasconcentration G1 of the interior of the incubator; maintaining the firsttemperature T1 and the CO₂ gas concentration G1 for a time t1; setting asecond temperature T2 and a second CO₂ gas concentration to a value G2;purging a portion of the gas concentration of the interior of theincubator via a purging mechanism for a time tp; and maintaining thesecond temperature T2 and the second CO₂ gas concentration G2 for a timet2.
 20. The method of claim 19, wherein setting a first temperature T1and a first CO₂ gas concentration G1 of the interior of the incubator issetting at the first temperature T1 to 37 degrees C. and the first CO₂gas concentration G1 to 5% CO₂; maintaining the first temperature T1 andthe CO₂ gas concentration G1 for a time t1 is for a time t1 of about 8hours; setting a second temperature T2 and a second CO₂ gasconcentration to a value G2 includes setting a second temperature 38degrees C. and a second CO₂ gas concentration G2 to 5.5% CO₂; purging aportion of the gas concentration of the interior of the incubator via apurging mechanism for a time tp includes for a time tp of about 1minute; and maintaining the second temperature T2 and the second CO₂ gasconcentration G2 for a time t2 includes for a time t2 of about 30minutes.
 21. The method of claim 19, wherein setting a first temperatureT1 and a first CO₂ gas concentration G1 of the interior of the incubatorincludes setting the first temperature T1 to 38 degrees C. and the firstCO₂ gas concentration G1 to 6% CO₂; maintaining the first temperature T1and the CO₂ gas concentration G1 for a time t1 is for a time t1 of about60 minutes; setting the second temperature T2 and the second CO₂ gasconcentration to a value G2 includes setting the second temperature to37 degrees C. and the second CO₂ gas concentration G2 to 5% CO₂; purginga portion of the gas concentration of the interior of the incubator viaa purging mechanism for a time tp includes for a time tp of about 1minute; and maintaining the second temperature T2 and the second CO₂ gasconcentration G2 for a time t2 includes for a time t2 of about 8 hours.22. The method of claim 19, setting a first temperature T1 and a firstCO₂ gas concentration G1 of the interior of the incubator includessetting at the first temperature T1 to 36 degrees C. and the first CO₂gas concentration G1 to 5.5% CO₂; maintaining the first temperature T1and the CO₂ gas concentration G1 for a time t1 is for a time t1 of about120 minutes; setting a second temperature T2 and a second CO₂ gasconcentration to a value G2 includes setting the second temperature to37 degrees C. and the second CO₂ gas concentration G2 to 5% CO₂; purginga portion of the gas concentration of the interior of the incubator viaa purging mechanism for a time tp includes for a time of about 1 minute;and maintaining the second temperature T2 and the second CO₂ gasconcentration G2 for a time t3 includes for a time t2 of about 20 hours.23. The method of claim 22, wherein executing at least one temperatureand gas concentration sequence for an interior of an incubator via aprogramming device further comprises: setting an O₂ gas concentrationlevel to a value G3 after setting the temperature T1 and the CO₂ gasconcentration value G1 via the programming device; maintaining each ofthe temperature T1, the CO₂ gas concentration value G1, and the O₂ gasconcentration value G3 for the time t1 via the programming device;setting the CO₂ gas concentration value to a value G4 via theprogramming device; setting the O₂ gas concentration value to a value G5via the programming device; and maintaining the CO₂ gas concentrationvalue to a value G4 and the O₂ gas concentration value to a value G5 fora period of time t1 via the programming device.
 24. The method of claim23, further comprising setting the O₂ gas concentration value to zerobefore operating the purging mechanism for a time tp via the programmingdevice.
 25. The method of claim 23, wherein at least one or more of theO₂ gas concentration value G3 is 18% O₂, the CO₂ gas concentration valueG4 is 6% CO₂, and the O₂ gas concentration value G5 is 15% O₂.
 26. Themethod of claim 16, wherein enabling rapid changes in at least onetemperature and gas concentration within the interior of the incubatorvia a purging mechanism coupled to the incubator and the programmingdevice comprises purging a portion of at least the CO₂ gas concentrationin the interior of the incubator via actuating the purging mechanism fora limited period of time.
 27. The method of claim 16, wherein enablingrapid changes in the temperature within the interior of the incubatorvia a purging mechanism coupled to the incubator and the programmingdevice comprises releasing an amount of water disposed in an internaltank of the incubator to enable rapid changes in the temperature of theincubator
 28. The method of claim 16, further comprising preventing lossof gas concentration of the interior of the incubator when the purgingmechanism is not operating via an air lock assembly.
 29. The method ofclaim 16, further comprising listing one or more of an actualtemperature of the interior of the incubator, an actual gasconcentration of the interior of the incubator, and at least one log ofa previous actual temperature and actual gas concentration over a periodof time on a display disposed on an outside surface of the incubator.30. The method of claim 16, further comprising repeating the temperatureand gas concentration sequence when the temperature and gasconcentration sequence is complete.